Unknown,Transcriptomics,Genomics,Proteomics

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Steroids_Effect_on_HUVEC_Response_to_LPS_or_Cytokines

ABSTRACT: Pooled first passage cryopreserved, human umbilical vein endothelial cells (HUVEC) were obtained from Cascade Biologics (Portland, OR), thawed, and cultured according to the supplierâs recommendations in Medium 200. After thawing, cells were seeded onto 0.2% gelatin coated tissue culture plates and cultivated under 95% air, 5% CO2 at 37 oC. Confluent cells were cultured in steroids-depleted medium for 96 hrs before the addition of 17Î²-estradiol (17Î²E) or hydrocortisone (H) to 1 ÂµM for 5 hrs. For Lipopolysaccharide (LPS) or cytokine mix (CM)-treatments, cells or pretreated with vehicle (0.1% ethanol), 1ÂµM 17Î²E, or H for 1 hr were exposed to 100 ng/ml LPS or a mixture of proinflammatory cytokines (CM, 500 U/ml IL-1b, 2500 U/ml TNF-Î±, and 1250 U/ml IFN-Î³) for 4 hours. All treatments were done in quadruplicates. At the end of treatment, total cellular RNA was isolated.

ORGANISM(S): Homo sapiens

SUBMITTER: Phillip Bowman

PROVIDER: E-GEOD-1486 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:It is now well established that mature mammalian spermatozoa carry a population of mRNA molecules, at least some of which are transferred to the oocyte at fertilisation. However, the function of the sperm transcriptome remains largely unclear. To shed light on the evolutionary conservation of this feature of sperm biology, we analysed highly purified populations of mature sperm from the fruitfly, Drosophila melanogaster. As with mammalian sperm, we found a consistently enriched population of mRNA molecules that are not likely to be derived from contaminating somatic cells or immature sperm. Using tagged transcripts for three of the spermatozoal mRNAs, we demonstrate that they are transferred to the oocyte at fertilisation and can be detected at least until the onset of zygotic gene expression. We find a remarkable conservation in the functional annotations associated with fly and human spermatozoal mRNAs, in particular a highly significant enrichment for transcripts encoding Ribosomal Proteins. The identification of a conserved set of spermatozoal transcripts opens the possibility of using the power of Drosophila genetics to address the function of this enigmatic class of molecules. RNA extracted from three biological replicates of purified sperm (Sperm rep1, Sperm rep2 and Sperm rep3) was used as a template for oligo-dT-primed reverse transcription, amplification, labelling of dye swapped technical replicates and hybridisation to long oligonucleotides microarrays. As a control, RNA from two biological replicates of dissected adult testis plus accessory glands (Testis_rep1, Testis_rep2) was amplified, labelled (dye-swap technical replicate) and hybridised to similar arrays. To help with the spot-finding of the arrays genomic DNA was co-hybridised in some cases (this genomic DNA data was excluded from further analysis). Genes with an intensity level below 200 in at least one channel across the Sperm or Testis set were removed (5579 transcripts present in all three sperm replicates, 5358 transcripts from the testis/accessory gland samples and 4295 transcripts common to both data sets). Then the quantile normalisation was independently applied to the Sperm replicate samples and Testis replicates.

Project description:Hypoxia-induced cell injury has been related to multiple pathological conditions. In order to render hypoxia-sensitive cells and tissues resistant to low O2 environment, in this current study, we used Drosophila melanogaster as a model to dissect the mechanisms underlying hypoxia-tolerance. A D. melanogaster strain that lives perpetually in an extremely low-oxygen environment (4% O2, an oxygen level that is equivalent to that over about 4,000 m above Mt. Everest) was generated through laboratory selection pressure using a continuing reduction of O2 over many generations. This phenotype is genetically stable since selected flies, after several generations in room air, survive at this low O2 level. Gene expression profiling showed striking differences between tolerant and naÃ¯ve flies, in larvae and adults, both quantitatively and qualitatively. Up-regulated genes in the tolerant flies included signal transduction pathways (e.g., Notch and Toll/Imd pathways), but metabolic genes were remarkably down-regulated in the larvae. Furthermore, a different allelic frequency and enzymatic activity of the triose phosphate isomerase (TPI) was present in the tolerant versus naÃ¯ve flies. The transcriptional suppressor, hairy, was up-regulated in the microarrays and its binding elements were present in the regulatory region of the specifically down-regulated metabolic genes but not others, and mutations in hairy significantly reduced hypoxia tolerance. We conclude that, the hypoxia-selected flies: (a) altered their gene expression and genetic code, and (b) coordinated their metabolic suppression, especially during development, with hairy acting as a metabolic switch, thus playing a crucial role in hypoxia-tolerance. Keywords: genetic bases of hypoxia adaptation 27 isogenic D. melanogaster Lines were pooled and following long-term selection over generations with decreased oxygen level in the culture environment. The differences in gene expression were compared between adapted flies and generation matched naive controls by microarray. Pooled RNA samples from 3rd instar larvae of 27 parental lines were used as common reference.

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Steroids_Effect_on_HUVEC_Response_to_LPS_or_Cytokines

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